The present invention relates generally to selected fused pyrrolocarbazoles, including pharmaceutical compositions thereof and methods of treating diseases therewith. The present invention is also directed to intermediates and processes for making these fused pyrrolocarbazoles.
Publications cited throughout this disclosure are incorporated in their entirety herein by reference.
Various synthetic small organic molecules that are biologically active and generally known in the art as xe2x80x9cfused pyrrolocarbazolesxe2x80x9d have been prepared (See U.S. Pat. Nos. 5,475,110; 5,591,855; 5,594,009; and 5,616,724). In addition, U.S. Pat. No. 5,705,511 discloses fused pyrrolocarbazole compounds which possess a variety of functional pharmacological activities. The fused pyrrolocarbazoles were disclosed to be used in a variety of ways, including: enhancing the function and/or survival of cells of neuronal lineage, either singularly or in combination with neurotrophic factor(s) and/or indolocarbozoles; enhancing trophic factor-induced activity; inhibition of protein kinase C (xe2x80x9cPKCxe2x80x9d) activity; inhibition of trk tyrosine kinase activity; inhibition of proliferation of a prostate cancer cell-line; inhibition of the cellular pathways involved in the inflammation process; and enhancement of the survival of neuronal cells at risk of dying.
The present inventors have found that certain selected fused pyrrolocarbazoles selected from the generic formulas of U.S. Pat. No. 5,705,511 but not specifically disclosed therein possess surprising and unexpected biological activities compared to the compounds described in U.S. Pat. No. 5,705,511.
Accordingly, one object of the invention is to provide novel fused pyrrolocarbazole compounds represented by the general Formula I: 
Constituent members of Formula I are disclosed in detail, infra.
Preferred fused pyrrolocarbazoles are represented by the following Formula II: 
Constituent members of Formula I are disclosed in detail, infra.
The fused pyrrolocarbazoles of the present invention may be used in a variety of ways, including: inhibition of angiogenesis; antitumor agents; enhancing the function and/or survival of cells of neuronal lineage, either singularly or in combination with neurotrophic factor(s) and/or indolocarbozoles; enhancing trophic factor-induced activity; inhibition of kinases; inhibition of vascular endothelial growth factor receptor (VEGFR) kinase, preferably VEGFR2; inhibition of mixed lineage kinase (MLK); trk kinase; inhibition of platelet derived growth factor receptor (PDGFR) kinase; inhibition of NGF-stimulated trk phosphorylation; inhibition of protein kinase C (xe2x80x9cPKCxe2x80x9d) activity; inhibition of trk tyrosine kinase activity; inhibition of proliferation of a prostate cancer cell-line; inhibition of the cellular pathways involved in the inflammation process; and enhancement of the survival of neuronal cells at risk of dying. In addition, the fused pyrrolocarbazoles may useful for inhibition of c-met, c-kit, and mutated Flt-3 containing internal tandem duplications in the juxtamembrane domain. Because of these varied activities, the disclosed compounds find utility in a variety of settings, including research and therapeutic environments.
Another object of the present invention is to provide pharmaceutical compositions comprising a fused pyrrolocarbazole of the present invention wherein the compositions comprise a pharmaceutically acceptable excipient or carrier and a therapeutically effective amount of at least one of the compounds of the present invention, or a pharmaceutically acceptable salt or ester form thereof.
It is another object of the present invention to provide methods of treating or preventing diseases or disorders comprising administering a therapeutic or preventative effective amount of at least one of the compounds of the present invention to a subject in need thereof.
These and other objects, features and advantages of the fused pyrrolocarbazoles will be disclosed in the following detailed description of the patent disclosure.
One embodiment of the present invention are the fused pyrrolocarbazoles represented by Formula I: 
wherein:
R1 and R2 are the same or different and are independently selected from H, or alkyl of 1-8 carbons (inclusive), preferably an alkyl of 1-4 carbons (inclusive), substituted with OH, or xe2x80x94OR4 where R4 is an alkyl of 1-4 carbons (inclusive), aryl, preferably phenyl or naphthyl, or the residue of an amino acid after the hydroxyl group of the carboxyl group is removed; and
R3 is xe2x80x94CH2OH; xe2x80x94CH2OR7; xe2x80x94(CH2)nSR5; xe2x80x94(CH2)nS(O)yR5; xe2x80x94CH2SR5; or alkyl of 1-8 carbons (inclusive), preferably an alkyl of 1-4 carbons (inclusive), substituted with xe2x80x94OH, xe2x80x94OR5, xe2x80x94OR8, xe2x80x94CH2OR7, xe2x80x94S(O)yR6 or SR6; and wherein
R5 is alkyl of 1-4 carbons (inclusive), or aryl, preferably phenyl or naphthyl;
R6is H, alkyl of 1-4 carbons (inclusive), aryl of 6-10 carbons, preferably phenyl or naphthyl, or heteroaryl;
R7 is H or alkyl of 1-4 carbons (inclusive);
R8 is the residue of an amino acid after the hydroxyl group of the carboxyl group is removed;
n is an integer of 1-4 (inclusive); and
y is 1 or 2.
In certain preferred embodiments, the compounds of Formula I are those of Formula II: 
wherein R1, R2, and R3 are as defined for Formula I above.
In certain referred embodiments, R1 is an alkyl of 1-4 carbons (inclusive), substituted with xe2x80x94OH or xe2x80x94OR4 where R4 is an alkyl of 1-4 carbons (inclusive), aryl, preferably phenyl or naphthyl, or the residue of an amino acid after the hydroxyl group of the carboxyl group is removed;
R2 is H; and R3 is xe2x80x94CH2OH; xe2x80x94CH2OR7; xe2x80x94(CH2)nSR5; xe2x80x94(CH2)nS(O)yR5; xe2x80x94CH2SR5; or alkyl of 1-8 carbons (inclusive), preferably an alkyl of 1-4 carbons (inclusive), substituted with xe2x80x94OH, xe2x80x94OR5, xe2x80x94OR8, xe2x80x94CH2OR7, xe2x80x94S(O)yR6 or SR6;
wherein R5, R6, R7 and R8 are as defined for Formula I above.
In certain other preferred embodiments, R1 is xe2x80x94CH2CH2CH2OH or xe2x80x94CH2CH2CH2OCOCH2N(CH3)2, R2 is H and R3 is xe2x80x94CH2OR7; wherein R7 is alkyl of 1-4 carbons (inclusive).
In certain even further preferred embodiments the fused pyrrolocarbazoles of Formula I and Formula II are those represented in Table I:
Preferred fused pyrrolocarbazoles of Formula II are represented structurally in Table II:
Particularly preferred compounds of Table II include compounds 1, 3, 4, 5, 6, 7 and 22 with compounds 3 and 22 being most preferred.
The compounds represented by Formula I and II and depicted in Tables I and II may also be referred to herein as xe2x80x9cthe compounds,xe2x80x9d xe2x80x9cthe compound(s) of the present invention,xe2x80x9d xe2x80x9cfused pyrrolocarbazole(s),xe2x80x9d xe2x80x9cfused pyrrolocarbazole(s) of the present inventionxe2x80x9d and the like.
Certain compounds of U.S. Pat. No. 5,705,511 are depicted in Table IIa.
As used herein with reference to the definitions of R1 and R2, the term xe2x80x9camino acidxe2x80x9d denotes a molecule containing both an amino acid group and a carboxyl group. It includes an xe2x80x9cxcex1-amino acidxe2x80x9d which has its usual meaning as a carboxylic acid which bears an amino functionality on the carbon adjacent to the carboxyl group. xcex1-Amino acids can be naturally occurring or non-naturally occurring. Amino acids also include xe2x80x9cdipeptidesxe2x80x9d which are defined herein as two amino acids which are joined in a peptide linkage. Thus constituents of dipeptides are not limited to xcex1-amino acids, and can be any molecule containing both an amino group and a carboxyl group. Preferred are xcex1-amino acids, dipeptides such as lysyl-xcex2-alanine, and aminoalkanoic acids of 2-8 carbons, e.g., 3-dimethylaminobutyric acid.
Pharmaceutically acceptable salts of the fused pyrrolocarbazoles of the present invention also fall within the scope of the compounds as disclosed herein. The term xe2x80x9cpharmaceutically acceptable saltsxe2x80x9d as used herein means an inorganic acid addition salt such as hydrochloride, sulfate, and phosphate, or an organic acid addition salt such as acetate, maleate, fumarate, tartrate, and citrate. Examples of pharmaceutically acceptable metal salts are alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt, and zinc salt. Examples of pharmaceutically acceptable ammonium salts are ammonium salt and tetramethylammonium salt. Examples of pharmaceutically acceptable organic amine addition salts are salts with morpholine and piperidine. Examples of pharmaceutically acceptable amino acid addition salts are salts with lysine, glycine, and phenylalanine.
Compounds provided herein can be formulated into pharmaceutical compositions by admixture with pharmaceutically acceptable nontoxic excipients or carriers. As noted above, such compositions may be prepared for use in parenteral administration, particularly in the form of liquid solutions or suspensions; or oral administration, particularly in the form of tablets or capsules; or intranasally, particularly in the form of powders, nasal drops, or aerosols; or dermally, via, for example, trans-dermal patches.
Accordingly, another aspect of the present invention are pharmaceutical compositions comprising a compound of the present invention optionally in admixture with one or more pharmaceutically acceptable excipients or carriers. Preferably, the pharmaceutical compositions comprise a compound of Formula II. More preferably, the pharmaceutical compositions comprise a compound of Table I or Table II.
In certain preferred pharmaceutical compositions, the composition is for inhibiting one or more of trk kinase activity, VEGFR kinase activity, PKC or PDGFR activity wherein the composition comprises a compound of Formula I, Formula II, Table I or Table II and optionally one or more pharmaceutically acceptable carrier(s).
In other preferred pharmaceutical compositions the composition is for enhancing tropic factor or spinal chord ChAT activity wherein the composition comprises a compound of Formula I, Formula II, Table I or Table II and a pharmaceutically acceptable carrier. In other preferred pharmaceutical compositions, the composition is for treating or preventing angiogenesis and angiogenic disorders such as cancer of solid tumors, endometriosis, retinopathy, diabetic retinopathy, psoriasis, hemangioblastoma, ocular disorders or macular degeneration. In other preferred pharmaceutical compositions, the composition is for treating or preventing neoplasia, rheumatoid arthritis, pulmonary fibrosis, myelofibrosis, abnormal wound healing, atherosclerosis, or restenosis. In other preferred pharmaceutical compositions, the composition is for treating or preventing neurodegenerative diseases and disorders, Alzheimer""s disease, amyotrophic lateral sclerosis, Parkinson""s disease, stroke, ischaemia, Huntington""s disease, AIDS dementia, epilepsy, multiple sclerosis, peripheral neuropathy, chemotherapy induced peripheral neuropathy, AIDS related peripheral neuropathy, or injuries of the brain or spinal chord. In other preferred pharmaceutical compositions, the composition is for treating or preventing prostate disorders such as prostate cancer or benign prostate hyperplasia. In still other preferred pharmaceutical compositions, the composition is used for treating or preventing multiple myeloma and leukemias including, but not. limited to, acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, and chronic lymphocytic leukemia.
The compositions may conveniently be administered in unit dosage form and may be prepared by any of the methods well known in the pharmaceutical art, for example, as described in Remington""s Pharmaceutical Sciences (Mack Pub. Co., Easton, Pa., 1980). Formulations for parenteral administration may contain as common excipients sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils and vegetable origin, hydrogenated naphthalenes and the like. In particular, biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be useful excipients to control the release of the active compounds. Other potentially useful parenteral delivery systems for these active compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation administration contain as excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally. Formulations for parenteral administration may also include glycocholate for buccal administration, a salicylate for rectal administration, or citric acid for vaginal administration. Formulations for trans-dermal patches are preferably lipophilic emulsions.
The compounds of the present invention can be employed as the sole active agent in a pharmaceutical or can be used in combination with other active ingredients, e.g., other growth factors which could facilitate neuronal survival or axonal regeneration in diseases or disorders or other angiogenesis or antitumor agents.
The concentrations of the compounds described herein in a therapeutic or pharmaceutical composition will vary depending upon a number of factors, including the dosage of the drug to be administered, the chemical characteristics (e.g., hydrophobicity) of the compounds employed, and the route of administration. In general terms, the compounds of this invention may be provided in an aqueous physiological buffer solution containing about 0.1 to 10% w/v compound for parenteral administration. Typical dose ranges are from about 1 xcexcg/kg to about 1 g/kg of body weight per day; a preferred dose range is from about 0.01 mg/kg to 100 mg/kg of body weight per day. The preferred dosage of drug to be administered is likely to depend on such variables as the type and extent of progression of the disease or disorder, the overall health status of the particular patient, the relative biological efficacy of the compound selected, and formulation of the compound excipient, and its route of administration.
In other embodiments, the present invention provides a method for inhibiting trk kinase activity comprising providing a compound of the present invention in an amount sufficient to result in effective inhibition. In a preferred embodiment, the compound of the present invention is provided to treat inflammation, for example, neurological inflammation and chronic arthritis inflammation. In another preferred embodiment, the trk kinase receptor is trk A.
In other embodiments, the present invention provides a method for treating or preventing prostate disorders which comprises administering to a host in need of such treatment or prevention a therapeutically effective amount of a compound of the present invention. In a preferred embodiment, the prostate disorder is prostate cancer or benign prostate hyperplasia.
In other embodiments, the present invention provides a method for treating or preventing angiogenic disorders where VEGFR kinase activity contributes to pathological conditions, the method comprising providing a compound of the present invention in an amount sufficient to result in the vascular endothelial growth factor receptor being contacted with an effective inhibitory amount of the compound. In another embodiment, the present invention provides a method for treating or preventing angiogenic disorders which comprises administering to a host in need of such treatment or prevention a therapeutically effective amount of a compound of the present invention. In a preferred embodiment, the angiogenic disorder is cancer of solid tumors, ocular disorders, macular degeneration, endometriosis, diabetic retinopathy, psoriasis, or hemangioblastoma.
In other embodiments, the present invention provides a method for treating or preventing disorders where PDGFR activity contributes to pathological conditions, the method comprising providing a compound of the present invention in an amount sufficient to result in the platelet derived growth factor receptor being contacted with an effective inhibitory amount of the compound. In another embodiment, the present invention provides a method for treating or preventing pathological disorders which comprises administering to a host in need of such treatment or prevention a therapeutically effective amount of a compound of the present invention. In preferred embodiments, the pathological disorder is neoplasia, rheumatoid arthritis, chronic arthritis, pulmonary fibrosis, myelofibrosis, abnormal wound healing, atherosclerosis, or restenosis.
In other embodiments, the present invention provides a method for treating disorders characterized by the aberrant activity of trophic factor responsive cells, the method comprising providing a compound of Formula I, Formula II, Table I or Table II in an amount sufficient to result in the trophic factor cell receptor being contacted with an effective activity inducing amount of the compound. In preferred embodiments, the activity of the trophic factor responsive cells is ChAT activity. In another embodiment, the present invention provides a method for treating or preventing neurodegenerative diseases and disorders, Alzheimer""s disease, amyotrophic lateral sclerosis, Parkinson""s disease, stroke, ischaemia, Huntington""s disease, AIDS dementia, epilepsy, multiple sclerosis, peripheral neuropathy, chemotherapy induced peripheral neuropathy, AID related peripheral neuropathy or injuries of the brain or spinal chord which comprises administering to a host in need of such treatment or prevention a therapeutically effective amount of a compound of Formula I, Formula II, Table I and Table II.
As used herein, the term xe2x80x9ceffectxe2x80x9d when used to modify the terms xe2x80x9cfunctionxe2x80x9d and xe2x80x9csurvivalxe2x80x9d means a positive or negative alteration or change. An effect which is positive can be referred to herein as an xe2x80x9cenhancementxe2x80x9d or xe2x80x9cenhancingxe2x80x9d and an effect which is negative can be referred to herein as xe2x80x9cinhibitionxe2x80x9d or xe2x80x9cinhibiting.xe2x80x9d
As used herein, the terms xe2x80x9cenhancexe2x80x9d or xe2x80x9cenhancingxe2x80x9d when used to modify the terms xe2x80x9cfunctionxe2x80x9d or xe2x80x9csurvivalxe2x80x9d means that the presence of a fused pyrrolocarbazole has a positive effect on the function and/or survival of a trophic factor responsive cell compared with a cell in the absence of the fused pyrrolocarbazole. For example, and not by way of limitation, with respect to the survival of, e.g., a cholinergic neuron, the fused pyrrolocarbazole would evidence enhancement of survival of a cholinergic neuronal population at risk of dying (due to, e.g., injury, a disease condition, a degenerative condition or natural progression) when compared to a cholinergic neuronal population not presented with such fused pyrrolocarbazole, if the treated population has a comparatively greater period of functionality than the non-treated population. As a further example, and again not by way of limitation, with respect to the function of, e.g., a sensory neuron, the fused pyrrolocarbazole would evidence enhancement of the function (e.g. neurite extension) of a sensory neuronal population when compared to a sensory neuronal population not presented with such fused pyrrolocarbazole, if the neurite extension of the treated population is comparatively greater than the neurite extension of the non-treated population.
As used herein, xe2x80x9cinhibitxe2x80x9d and xe2x80x9cinhibitionxe2x80x9d mean that a specified response of a designated material (e.g., enzymatic activity) is comparatively decreased in the presence of a fused pyrrolocarbazole of the present invention.
As used herein the term xe2x80x9cneuron,xe2x80x9d xe2x80x9ccell of neuronal lineagexe2x80x9d and xe2x80x9cneuronal cellxe2x80x9d includes, but is not limited to, a heterogeneous population of neuronal types having singular or multiple transmitters and/or singular or multiple functions; preferably, these are cholinergic and sensory neurons. As used herein, the phrase xe2x80x9ccholinergic neuronxe2x80x9d means neurons of the Central Nervous System (CNS) and Peripheral Nervous System (PNS) whose neurotransmitter is acetylcholine; exemplary are basal forebrain and spinal cord neurons. As used herein, the phrase xe2x80x9csensory neuronxe2x80x9d includes neurons responsive to environmental cues (e.g., temperature, movement) from, e.g., skin, muscle and joints; exemplary is a neuron from the DRG.
As used herein a xe2x80x9ctrophic factorxe2x80x9d is a molecule that directly or indirectly affects the survival or function of a trophic factor responsive cell. Exemplary trophic factors include Ciliary Neurotrophic Factor (CNTF), basic Fibroblast Growth Factor (bFGF), insulin and insulin-like growth factors (e.g., IGF-I, IGF-II, IGF-III), interferons, interleukins, cytokines, and the neurotrophins, including Nerve Growth Factor (NGF), Neurotrophin-3 (NT-3), Neurotrophin-4/5 (NT-4/5) and Brain Derived Neurotrophic Factor (BDNF).
A xe2x80x9ctrophic factor-responsive cell,xe2x80x9d as defined herein, is a cell which includes a receptor to which a trophic factor can specifically bind; examples include neurons (e.g., cholinergic and sensory neurons) and non-neuronal cells (e.g., monocytes and neoplastic cells).
As used herein, xe2x80x9ctrophic factor activityxe2x80x9d and xe2x80x9ctrophic factor induced activityxe2x80x9d are defined as any response which directly or indirectly results from the binding of a trophic factor (e.g., NGF) to a cell comprising a trophic factor receptor (e.g., neuron comprising of a trk). In the case of, e.g., NGF binding with trk, an exemplary response would include autophosphorylation of trk tyrosine residues leading to increased ChAT activity which results in enhanced neuron survival, and/or function.
As used in the phrases xe2x80x9ctrophic factor activityxe2x80x9d and xe2x80x9ctrophic factor-induced activity,xe2x80x9d the term xe2x80x9ctrophic factorxe2x80x9d includes both endogenous and exogenous trophic factors, where xe2x80x9cendogenousxe2x80x9d refers to a trophic factor normally present and xe2x80x9cexogenousxe2x80x9d refers to a trophic factor added to a system. As defined, xe2x80x9ctrophic factor induced activityxe2x80x9d includes activity induced by (1) endogenous trophic factors; (2) exogenous trophic factors; and (3) a combination of endogenous and exogenous trophic factors.
As used herein, the term xe2x80x9ctrkxe2x80x9d refers to the family of high affinity neurotrophin receptors presently comprising trk A, trk B and trk C, and other membrane associated proteins to which a neurotrophin can bind.
As used herein the phrase xe2x80x9chyperproliferative statexe2x80x9d in reference to the term xe2x80x9ccellsxe2x80x9d means cells whose unregulated and/or abnormal growth can lead to the development of an unwanted condition, for example, a cancerous condition or a psoriatic condition.
As used herein, xe2x80x9ccancerxe2x80x9d and xe2x80x9ccancerousxe2x80x9d refer to any malignant proliferation of cells in a mammal. Examples include prostate, benign prostate hyperplasia, ovarian, breast and other recognized cancers. As used herein the term xe2x80x9cpsoriasisxe2x80x9d and xe2x80x9cpsoriatic conditionxe2x80x9d refer to disorders involving keratinocyte hyperproliferation, inflammatory cell infiltration and cytokine alteration.
As used herein, the phrase xe2x80x9cat risk of dyingxe2x80x9d in conjunction with a biological material, e.g., a cell such as a neuron, means a state or condition which negatively impacts the biological material such that the material has an increased likelihood of dying due to such state or condition. For example, compounds disclosed herein can xe2x80x9crescuexe2x80x9d or enhance the survival of motoneurons which are naturally at risk of dying in an in ovo model of programmed cell death. Similarly, for example, a neuron may be at risk of dying due to the natural aging process which occasions the death of a neuron, or due to an injury, such as a trauma to the head, which may be such that neurons and/or glia, for example, impacted by such trauma may be at risk of dying. Further, for example, a neuron may be at risk of dying due to a disease state or condition, as in the case of neurons at risk of dying as occasioned by the disease ALS. Thus, by enhancing the survival of a cell at risk of dying by use of a compound of the claimed invention is meant that such compound decreases or prevents the risk of the death of the cell.
As used herein the term xe2x80x9ccontactingxe2x80x9d means directly or indirectly causing placement together of moieties, such that the moieties directly or indirectly come into physical association with each other, whereby a desired outcome is achieved. Thus, as used herein, one can xe2x80x9ccontactxe2x80x9d a target cell with a compound as disclosed herein even though the compound and cell do not necessarily physically join together (as, for example, is the case where a ligand and a receptor physically join together), as long as the desired outcome is achieved (e.g., enhancement of the survival of the cell). Contacting thus includes acts such as placing moieties together in a container (e.g., adding a compound as disclosed herein to a container comprising cells for in vitro studies) as well as administration of the compound to a target entity (e.g., injecting a compound as disclosed herein into a laboratory animal for in vivo testing, or into a human for therapy or treatment purposes).
As used herein, xe2x80x9cprodrugxe2x80x9d is intended to include any covalently bonded carriers which release the active parent drug as a compound of the present invention in vivo when such prodrug is administered to a mammalian subject. Since prodrugs are known to enhance numerous desirable qualities of pharmaceuticals (e.g., solubility, bioavailability, manufacturing, etc.) the compounds of the present invention may be delivered in prodrug form. Thus, the present invention contemplates prodrugs of the compounds of the present invention, compositions containing the same, and methods of treating diseases and disorders with such prodrugs. Prodrugs of a compound of the present invention, for example Formula I, may be prepared by modifying functional groups present in the compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compound. Accordingly, prodrugs include, for example, compounds of the present invention wherein a hydroxy, amino, or carboxy group is bonded to any group that, when the prodrug is administered to a mammalian subject, cleaves to form a free hydroxyl, free amino, or carboxylic acid, respectively. Examples include, but are not limited to, the residue of an amino acid after the hydroxyl group of the carboxyl group is removed acetate, formate and benzoate derivatives of alcohol and amine functional groups; and alkyl, carbocyclic, aryl, and alkylaryl esters such as methyl, ethyl, propyl, iso-propyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, phenyl, benzyl, and phenethyl esters, and the like.
The fused pyrrolocarbazoles of the present invention have important functional pharmacological activities which find utility in a variety of settings, including both research and therapeutic arenas. For ease of presentation, and in order not to limit the range of utilities for which these compounds can be characterized, we generally describe the activities of the fused pyrrolocarbazoles as follows:
1. Inhibition of enzymatic activity
2. Effect on the function and/or survival of trophic factor responsive cells
3. Inhibition of inflammation-associated responses
4. Inhibition of cell growth associated with hyperproliferative states
5. Inhibition of developmentally programmed motoneuron death
Inhibition of enzymatic activity can be determined using, for example, VEGFR inhibition (e.g., VEGFR2 inhibition), MLK inhibition (e.g., MLK1, MLK2 or MLK3 inhibition), PDGFR kinase inhibition, NGF-stimulated trk phosphorylation, PKC inhibition, or trk tyrosine kinase inhibition assays. Effect on the function and/or survival of trophic factor responsive cells, e.g., cells of a neuronal lineage, can be established using any of the following assays: (1) cultured spinal cord choline acetyltransferase (xe2x80x9cChATxe2x80x9d) assay; (2) cultured dorsal root ganglion (xe2x80x9cDRGxe2x80x9d) neurite extension assay; (3) cultured basal forebrain neuron (xe2x80x9cBFNxe2x80x9d) ChAT activity assay. Inhibition of inflammation-associated response can be established using an indoleamine 2,3-dioxygenase (xe2x80x9cIDOxe2x80x9d) mRNA assay. Inhibition of cell growth associated with hyperproliferative states can be determined by measuring the growth of cell lines of interest, such as an AT2 line in the case of prostate cancer. Inhibition of developmentally programmed motoneuron death can be assessed in ovo using embryonic chick somatic motoneurons, which cells undergo naturally occurring death between embryonic days 6 and 10, and analyzing inhibition of such naturally occurring cell death as mediated by the compounds disclosed herein.
The inhibition of enzymatic activity by the fused pyrrolocarbazole compounds of the present invention can be determined using, for example, the following assays:
VEGFR Inhibition Assay
MLK Inhibition Assay
PKC Activity Inhibition Assay
trkA Tyrosine Kinase Activity Inhibition Assay
Inhibition of NGF-stimulated trk phosphorylation in a whole cell preparation
Platelet Derived Growth Factor Receptor (PDGFR) inhibition assay
Particularly, inhibition of the Vascular Endothelial Growth Factor Receptor (VEGFR) implies utility in, for example, diseases where angiogenesis plays important roles, such as cancer of solid tumors, endometriosis, diabetic retinopathy, psoriasis, hemangioblastoma, as well as other ocular diseases and cancers. Inhibition of MLK implies utility in, for example, neurological diseases. Inhibition of trk implies utility in, for example, diseases of the prostate such as prostate cancer and benign prostate hyperplasia, and treatment of inflammatory pain. Inhibition of the Platelet Derived Growth Factor Receptor (PDGFR) implies utility in, for example, various forms of neoplasia, rheumatoid arthritis, pulmonary fibrosis, myelofibrosis, abnormal wound healing, diseases with cardiovascular end points, such as atherosclerosis, restenosis, post-angioplasty restenosis, and the like.
Fused pyrrolocarbazoles have also been shown to have positive effects on the function and survival of trophic factor responsive cells by promoting the survival of neurons. With respect to the survival of a cholinergic neuron, for example, the compound may preserve the survival of a cholinergic neuronal population at risk of dying (due to, e.g., injury, a disease condition, a degenerative condition or natural progression) when compared to a cholinergic neuronal population not presented with such compound, if the treated population has a comparatively greater period of functionality than the non-treated population.
A variety of neurological disorders are characterized by neuronal cells which are dying, injured, functionally compromised, undergoing axonal degeneration, at risk of dying, etc. These disorders include, but are not limited to, neurological diseases and disorders, Alzheimer""s disease; motor neuron disorders (e.g. amyotrophic lateral sclerosis); Parkinson""s disease; cerebrovascular disorders (e.g., stroke, ischaemia); Huntington""s disease; AIDS dementia; epilepsy; multiple sclerosis; peripheral neuropathies (e.g., those affecting DRG neurons in chemotherapy-associated peripheral neuropathy) including diabetic neuropathy and AIDS related peripheral neuropathy; disorders induced by excitatory amino acids; and disorders associated with concussive or penetrating injuries of the brain or spinal cord.
The compounds are not only useful for enhancing trophic factor-induced activities of trophic responsive cells, e.g., cholinergic neurons, but also may function as survival promoting agents for other neuronal cell types, e.g., dopaminergic or glutamatergic. Growth factor may regulate survival of neurons by signaling cascades downstream of the small GTP binding proteins ras, rac, and cdc42 (Denhardt, D. T., Biochem. J., 1996, 318, 729). Specifically, activation of ras leads to phosphorylation and activation of extracellular receptor-activated kinase (ERK), which has been linked to biological growth and differentiation processes.
Stimulation of rac/cdc42 leads to an increase in activation of JNK and p38, responses that are associated with stress, apoptosis, and inflammation. Although growth factor responses are primarily via the ERK pathway, affecting these latter processes may lead to alternative mechanisms of neuronal survival which may mimic growth factor enhancing survival properties (Xia et al., Science, 1995, 270, 1326). The compounds of the present invention may also function as survival promoting agents for neuronal and non-neuronal cells by mechanisms related to, but also distinct from, growth factor mediated survival, for example, inhibition of the JNK and p38 MAPK pathways which may lead to survival by inhibition of apoptotic cell death processes.
The present compounds are also useful in the treatment of disorders associated with decreased ChAT activity or the death, injury to spinal cord motoneurons, and also have utility in, for example, diseases associated with apoptotic cell death of the central and peripheral nervous system, immune system and in inflammatory diseases. ChAT catalyzes the synthesis of the neurotransmitter acetylcholine, and it is considered an enzymatic marker for a functional cholinergic neuron. A functional neuron is also capable of survival. Neuron survival is assayed by quantitation of the specific uptake and enzymatic conversion of a dye (e.g., calcein AM) by living neurons. The compounds described herein may also find utility in the treatment of disease states involving malignant cell proliferation, such as many cancers.
Because of their varied utilities, the properties of isomeric fused pyrrolocarbazoles and isoindolones may be exploited in other settings, such as research. For example, the compounds can be used in the development of in vitro models of neuronal cell survival, function, identification, or for the screening of other synthetic compounds which have activities similar to that of the of isomeric fused pyrrolocarbazole and isoindolone compounds. Thus, the compounds provided by this invention are useful as standard or reference compounds for use in tests or assays for determining the activity of an agent in a pharmaceutical research program.
The compounds can also be utilized to investigate, define and determine molecular targets associated with functional responses. For example, by radiolabelling an isomeric fused pyrrolocarbazole or isoindolone compound associated with a specific cellular function (e.g., mitogenesis), the target entity to which the derivative binds can be identified, isolated, and purified for characterization. By way of further illustration, compounds may be used in the development of assays and models for further enhancement of the understanding of the roles that inhibition of serine/threonine or tyrosine protein kinase (e.g., PKC, trk tyrosine kinase) play in the mechanistic aspects of the associated disorders and diseases. Thus, the compounds of the present invention are useful as diagnostic reagents in diagnostic assays, such as the assays described herein.
The results obtained in the VEGFR and MLK assays are set forth below. Other assays are described in more detail as well. They are not intended, nor are they to be construed, as limiting the scope of the disclosure. Certain abbreviations used to delineate the results below are defined as follows: xe2x80x9cxcexcgxe2x80x9d denotes microgram, xe2x80x9cmgxe2x80x9d denotes milligram, xe2x80x9cgxe2x80x9d denotes gram, xe2x80x9cxcexcLxe2x80x9d denotes microliter, xe2x80x9cmLxe2x80x9d denotes milliliter, xe2x80x9cLxe2x80x9d denotes liter, xe2x80x9cnMxe2x80x9d denotes nanomolar, xe2x80x9cxcexcMxe2x80x9d denotes micromolar, xe2x80x9cmMxe2x80x9d denotes millimolar, xe2x80x9cMxe2x80x9d denotes molar and xe2x80x9cnmxe2x80x9d denotes nanometer.
Synthesis
The present invention also provides a method for preparing the fused pyrrolocarbazoles of the present invention. The compounds of the present invention may be prepared in a number of ways well known to those skilled in the art. The compounds can be synthesized, for example, by the methods described in the Schemes below, or variations thereon as appreciated by the skilled artisan. The appropriate modifications and substitutions being readily apparent and well known or readily obtainable from the scientific literature to those skilled in the art. All processes disclosed in association with the present invention are contemplated to be practiced on any scale, including milligram, gram, multigram, kilogram, multikilogram or commercial industrial scale.
It will be appreciated that the compounds of the present invention may contain one or more asymmetrically substituted carbon atoms, and may be isolated in optically active or racemic forms. Thus, all chiral, diastereomeric, racemic forms and all geometric isomeric forms of a structure are intended, unless the specific stereochemistry or isomeric form is specifically indicated. It is well known in the art how to prepare such optically active forms. For example, mixtures of stereoisomers may be separated by standard techniques including, but not limited to, resolution of racemic forms, normal, reverse-phase, and chiral chromatography, preferential salt formation, recrystallization, and the like, or by chiral synthesis either from active starting materials or by deliberate chiral synthesis of target centers.
As will be readily understood, functional groups present on the compounds of the present invention may contain protecting groups. For example, the amino acid side chain substituents of the compounds can be substituted with protecting groups such as benzyloxycarbonyl or t-butoxycarbonyl groups. Protecting groups are known per se as chemical functional groups that can be selectively appended to and removed from functionalities, such as hydroxyl groups and carboxyl groups. These groups are present in a chemical compound to render such functionality inert to chemical reaction conditions to which the compound is exposed. Any of a variety of protecting groups may be employed with the present invention. Preferred protecting groups include the benzyloxycarbonyl (Cbz; Z) group and the tert-butyloxycarbonyl (Boc) group. Other preferred protecting groups according to the invention may be found in Greene, T. W. and Wuts, P. G. M., xe2x80x9cProtective Groups in Organic Synthesisxe2x80x9d 2d. Ed., Wiley and Sons, 1991. 
Description of Synthesis
Compounds A and B were prepared by alkylation""s of indole I with 2-bromoethyl benzyl ether (A) or with 3-bromopropyl benzyl ether (B) using NaH in DMF, followed by debenzylation (Pd(OH)2/H2) as described in U.S. Pat. No. 5,705,511. Reference compound C was prepared by coupling B with Boc-leucine followed by deprotection of the BOC group using standard procedures know to those skilled in the art of organic synthesis. Compound B may also be prepared by reduction of ester IV with reducing agents such as LiBH4 followed by removal of the benzhydrol protecting group. The routes to prepare the benzyl ethers and thiol ethers are outlined in the schemes. Two methods are used to prepare the 3-hydroxymethyl intermediates 28-30, 33-35. Scheme 1 (Method A) delineates a carbonylation route while scheme 2 (method B) utilizes a formylation method. In scheme 1 Michael reaction of I with ethyl acrylate and a base such as DBU produces II, followed by lactam nitrogen protection with dimethoxybenzhydrol to III. Reduction of the ethyl ester using reducing agents such as lithium borohydride, followed by bromination with N-bromo succinimide provided intermediate V in good overall yield. Palladium catalyzed carbonylation of V in methoxyethanol gave the methoxyethoxy ester VI. After deprotection to VII, the ester could be reduced with reducing agents, for example diisobutylaluminum hydride (DIBAL-H) to give the diol 29. The formylation route to the hydroxymethyl compounds (method B, scheme 2) uses for example, HMTA in TFA or xcex1,xcex1-dichloromethyl methyl ether and a Lewis acid. The aldehydes may be reduced to hydroxymethyl compounds using reducing agents such as sodium borohydride or diisobutylaluminum hydride. The methyl ether or thio ether examples may be prepared using a general procedure outlined in scheme 4. In one approach, for example, the diol 29 may be converted to a tri-trifluoroacetate intermediate with trifluoroacetic anhydride and a base such as triethylamine, followed by treating this intermediate with an appropriate alkyl alcohol or alkyl thiol to give the benzyl ether (1-25, 27, 31, 32, 36-40) directly. In certain cases the trifluoroacetate ester of the primary alcohol may be isolated. In these examples the alcohol may be isolated by treatment of the trifluoroacetate with a base such as lithium hydroxide. In another approach, the ethers and thio ethers may be prepared by reacting a diol, for example 28 or 29, with an alcohol and an acid catalyst, such as p-toluene sulfonic acid or camphorsulfonic acid in a solvent, for example, methylene chloride, toluene or 1,2-dichloroethane.
Alcohols 33-35 were used to prepare ether examples 10-13, 15, 16 and 36. Examples 33-35 were prepared from ketones XI and XII as outlined in scheme 3. The ethers and thio ethers were prepared using the procedures described previously and outlined in scheme 4.
Example 7 was prepared as shown in scheme 5. Example 31 was alkylated with mesyl glycidol to give compound XIII. Reduction with triethylborohydride in THF produced example secondary alcohol 7. Example 14 was prepared (scheme 6) by treatment of compound XIV with cesium carbonate and acetaldehyde in methylene chloride/methanol. Example 40 was prepared as shown in scheme 7. The Di-TBS protected XVIII, was alkylated with paraformaldehyde using triton B/pyridine, followed by deprotection using TMSCl to give example 40. The amino acid esters, example 18-23 were prepared from example 3 and the corresponding carboxylic acid using standard coupling reaction known to those skilled in the art of organic synthesis.
Other features of the invention will become apparent in the course of the following descriptions of exemplary embodiments. These examples are given for illustration of the invention and are not intended to be limiting thereof.